Methods and compositions for peptide and protein labeling

ABSTRACT

The invention provides compositions and methods of use thereof for labeling peptide and proteins in vitro or in vivo. The methods described herein employ biotin ligase mutants and biotin analogs recognized by such mutants.

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. 119(e) to U.S.Provisional Patent Application No. 60/438,939, filed Jan. 9, 2003,entitled “SITE-SPECIFIC LABELING OF RECOMBINANT PROTEINS IN LIVING CELLSWITH ENGINEERED FLUOROPHORE TRANSFERASE”, the entire contents of whichare incorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made in part with government support under grantnumber K22-HG002671-01 from the National Institutes of Health. TheGovernment may retain certain rights in the invention.

BACKGROUND OF THE INVENTION

To track protein expression, localization, or conformational changes ascomponents of cellular signaling pathways, biologists need general toolsfor the in vivo site-specific labeling of proteins with fluorophores orother useful probes. Traditional chemical methods rely on thenucleophilicity of cysteine or lysine side chains and are toopromiscuous for in vivo use, and genetic methods such as fusion to greenfluorescent protein (GFP) carry bulky payloads (GFP is 238 amino acids)and are limited in the color range and nature of the spectroscopicreadout.

A survey of the existing methods for targeting small molecules toprotein sequences reveals that the shorter the target sequence, the lessspecific the conjugation chemistry. For instance, very specificconjugation can be achieved by fusing the protein O⁶-alkylguanine-DNAalkyltransferase (AGT) to the target protein of interest, and thenadding a fluorescently-labeled O⁶-benzylguanine suicide substrate forthe AGT. (Keppler, A. et al. Nat. Biotechnol. 21, 86–89, 2003). However,the AGT tag is 207 amino acids and introduces a large amount of stericbulk. Smaller peptide tags are more desirable, but difficult to targetwith small molecules with high specificity. For example, cysteinelabeling is not at all specific inside cells, and tetracysteine labeling(Griffin, B A et al. Science 281, 269–272, 1998), while much better, isstill insufficiently specific for most applications and allows only asmall set of probes to be introduced. Transglutaminase is already usedto label glutamine side chains with fluorophores in vitro (Sato, H. etal. Biochemistry 35, 13072–13080, 1996), however it is relativelypromiscuous for peptide and protein substrates, precluding its use inmammalian cells. In vitro labeling and microinjection has thedisadvantage that protein localization and abundance may be altered.Polyhistidine tag methodology has the disadvantage that nickel is toxic,promiscuous, membrane impermeant and a quencher of fluorescence.

Accordingly, there exists a need for a method to label proteins andpeptides that is specific and which offers a variety of a labelingoptions.

SUMMARY OF THE INVENTION

The invention relates in part to labeling of proteins (or fragmentsthereof) using biotin ligase mutants. The methods and compositionsprovided by the invention provide labeling specificity while alsoexpanding the scope of compatible probe structures for labeling ofproteins. Labeling of peptides or proteins can be performed in vitro orin vivo. The invention also provides, inter alia, biotin ligase mutantsand biotin analogs and methods of use thereof for labeling proteins. Italso provides screening methods for identifying further biotin ligasemutants and biotin analogs.

Thus, in one aspect, the invention provides a method for labeling atarget protein comprising contacting a fusion protein with a biotinanalog, and allowing sufficient time for the biotin analog to beconjugated to the fusion protein via an acceptor peptide, in thepresence of a biotin ligase mutant, wherein the fusion protein is afusion of the target protein and the acceptor peptide.

Various embodiments apply equally to this and other aspects of theinvention. These are discussed below.

In one embodiment, the biotin analog may comprise an aliphaticcarboxylic acid tail. In another embodiment and potentiallyadditionally, the biotin analog may comprise an amino acid substitutionat a trans-ureido nitrogen (N) of biotin. Examples of biotin analogsinclude but are not limited to N-ketone biotin analog, a ketone biotinanalog, an N-azide biotin analog, an azide biotin analog, an N-acylazide biotin analog, an NBD-GABA biotin analog, a 1,2-diamine biotinanalog, an N-alkyne biotin analog and a tetrathiol biotin analog. Thebiotin analog may be fluorogenic. Alternatively, the biotin analog maybe directly detectable. Examples include but are not limited tocoumarin, fluorescein, rhodamine, rosamine, an Alexa™ dye, resorufin,oregon green, tetramethyl rhodamine, Texas Red® and BODIPY. In stillother embodiments, the biotin analog is labeled with a directlydetectable label, such as but not limited to fluorophore, aradioisotope, a contrast agent, an MRI contrast agent, a PET label, aphosphorescent label and a luminescent label. Alternatively, the biotinanalog is labeled with an indirectly detectable label such as but notlimited to an enzyme, an enzyme substrate, an antibody, an antibodyfragment, an antigen, a hapten, a ligand, an affinity molecule, achromogenic substrate, a protein, a peptide, a nucleic acid, acarbohydrate and a lipid. In still a further embodiment, the biotinanalog is labeled with a membrane impermeant label.

The biotin analog may be labeled before or after conjugation to thefusion protein. In one embodiment, the acceptor peptide is fused to thetarget protein via a cleavable bond or linker.

The biotin analog may be labeled with a variety of labels, describedherein. For example, the biotin analog may be labeled with a singletoxygen radical generator such as but not limited to resorufin, malachitegreen, fluorescein or diaminobenzidine. The biotin analog may be labeledwith an analyte-binding group, such as a metal chelator, non-limitingexamples of which include EDTA, EGTA, a pyridinium, an imidazole and athiol. The biotin analog may be labeled with a heavy atom carrier, suchas but not limited to iodine. The biotin analog may be labeled with anaffinity tag such as but not limited to a histidine tag, a GST tag, aFLAG tag and an HA tag. The biotin analog may be labeled with aphotoactivatable cross-linker such as but not limited to benzophenonesand aziridines. The biotin analog may be labeled with a photoswitchlabel such as but not limited to azobenzene. The biotin analog may belabeled with a photolabile protecting group such as but not limited to anitrobenzyl group, a dimethoxy nitrobenzyl group or NVOC. The biotinanalog may be labeled with a peptide comprising non-naturally occurringamino acids, examples of which are provided herein.

The target protein may be a cell surface protein, or an intracellularprotein but it is not so limited. In one embodiment, the fusion proteinis in a cell. Depending upon the method, the biotin ligase mutant may beexpressed by a cell (for example the cell harboring the fusion protein)or it may be added to a protein in a cell free environment. In oneembodiment, the cell is a eukaryotic cell while in another it is abacterial cell. Examples of eukaryotic cell include but are not limitedto a mammalian cell, a Drosophila cell, a Zebrafish cell, a Xenopuscell, a yeast cell or a C. elegans cell.

In one embodiment, the acceptor peptide comprises an amino acid sequenceof SEQ ID NO: 4. In another embodiment, the acceptor peptide comprisesan amino acid sequence of SEQ ID NO: 5. The acceptor peptide may be N-or C-terminally fused to the target protein.

In still another embodiment, the biotin ligase mutant has an amino acidsubstitution at 83, 89, 90, 91, 92, 107, 112, 115, 116, 117, 118, 123,132, 134, 142, 186, 188, 189, 190, 204, 206, 207 or 235. In someembodiments, the amino acid substitution is at T90, C107, Q112, G115,Y132, S134, V189 or I207. In some important embodiments, the amino acidsubstitution is at T90 and includes but is not limited to T90G, T90A andT90V. In a particular embodiment, the amino acid substitution is at T90Gand optionally the biotin analog is N-ketone biotin analog. The biotinligase mutant may further comprise an amino acid substitution at N91such as but not limited to N91S, N91G, N91A or N91L. In a particularembodiment, the biotin ligase mutant comprises amino acid substitutionsof T90G and N91S. In a related embodiment, the biotin analog is N-alkynebiotin analog. In still other embodiments, the biotin ligase mutantcomprises amino acid substitutions of T90G/N91G, T90A/N90A or T90A/N91L.In still other embodiments, the amino acid substitution is C107G, Q112M,G115A, Y132G, Y132A, S134G, V189G or I207S. The biotin ligase mutant mayhave an amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 7.

The method may be performed in a cell free environment or it may beperformed in the context of a cell (e.g., in a cell or on a cell). Themethod may also be performed in a subject.

In another aspect, the invention provides a composition comprising abiotin ligase mutant that binds to a biotin analog. In one embodiment,the biotin ligase mutant comprises an amino acid substitution in abiotin interaction and activation domain. All of the foregoingembodiments relating to biotin ligase mutants and biotin analogs alsoapply to this aspect of the invention and thus will not be recitedagain. In another embodiment, the biotin ligase mutant is isolated. Thebiotin ligase mutant may have reduced binding affinity to biotin. Inanother embodiment, the biotin ligase mutant has wild type bindingaffinity to biotin.

In still another aspect, the invention provides a composition comprisinga nucleic acid encoding a biotin ligase mutant comprising an amino acidsubstitution at 83, 89, 90, 91, 92, 107, 112, 115, 116, 117, 118, 123,132, 134, 142, 186, 188, 189, 190, 204, 206, 207 or 235. As used herein,the amino acid positions recited herein are relative to the wild typebiotin ligase having an amino acid sequence as shown in SEQ ID NO:2. Itis to be understood that the biotin ligase mutant may comprise one ormore of the aforementioned amino acid substitutions. In particularembodiments, the amino acid substitution is selected from the groupconsisting of T90G, T91A, T90V, N91S, N91G, N91A, N91L, C 107G, Q112M,Q112G, G115A, Y132G, Y132A, S134G, V189G, and I207S. The nucleic acid ispreferably isolated, but it is not so limited. In some embodiments, thenucleic acid is inducibly expressed. The nucleic acid may encode any ofthe biotin ligase mutants described herein. The invention furtherprovides vectors that comprise nucleic acid that encode any of thebiotin ligase mutants described herein and host cells that comprisethese vectors. The invention further provides a process for preparing abiotin ligase mutant comprising culturing the host cells describedherein and recovering the biotin ligase mutant from the culture.

In yet another aspect, the invention provides a composition comprising abiotin analog that binds to a biotin ligase mutant, wherein the biotinanalog is alkyated at a trans-ureido nitrogen (N) of biotin. Examples ofsuch biotin analogs include but are not limited to an N-ketone biotinanalog, an N-azide biotin analog, an N-acyl azide biotin analog, and anN-alkyne biotin analog. In one embodiment, the biotin analog is notrecognized by wild type biotin ligase. In another embodiment, the biotinanalog is isolated. Other embodiments relating to biotin analogs andbiotin ligase mutants are recited herein.

In another aspect, the invention provides a composition comprising abiotin analog that binds to a biotin ligase mutant, wherein the biotinanalog is ketone biotin analog or NBD-GABA.

In still another aspect, the invention provides a phage display librarycomprising a biotin ligase mutant having an amino acid substitution at83, 89, 90, 91, 92, 107, 112, 115, 116, 117, 118, 123, 132, 134, 142,186, 188, 189, 190, 204, 206, 207 or 235. In one embodiment, the aminoacid substitution is at T90, G115, Y132, C107, Q112, V189, I207 or S134.In another embodiment, the amino acid substitution is at T90 and may bebut is not limited to T90G, T90A or T90V. In another embodiment, thebiotin ligase mutant further comprises an amino acid substitution at N91such as but not limited to N91S, N91G, N91A or N91L. In one embodiment,the biotin ligase mutant comprises amino acid substitutions of T90G andN91S. In another embodiment, it comprises one or more of the amino acidsubstitutions of C107G, Q112M, G115A, Y132G, Y132A, V189G, S134G, I207S,T90G/N91G, T90A/N91A and T90A/N91L. The amino acid substitution may beat 90, 91, 112, 115, 116, 132 or 188. In a particular embodiment, thelibrary has at least about 1×10⁸ or about 1×10⁹ members.

In still another aspect, the invention provides a method for identifyinga biotin ligase mutant having specificity for a biotin analog comprisingcontacting a biotin analog with an acceptor peptide in the presence of acandidate biotin ligase mutant molecule, and detecting a biotin analogthat is bound to the acceptor peptide, wherein the presence of thebiotin analog bound to the acceptor peptide indicates that the candidatebiotin ligase mutant molecule is a biotin ligase mutant havingspecificity for the biotin analog. The candidate molecule may be alibrary member such as but not limited to a phage display librarymember. In one embodiment, the candidate molecule is bound to a solidsupport while in another it is soluble. Various embodiments of biotinanalog are possible as recited herein. In one embodiment, detecting abiotin analog comprises detecting the detectable label conjugated to thebiotin analog. The acceptor peptide may have an amino acid sequencecomprising SEQ ID NO: 4 or SEQ ID NO: 5, but it is not so limited. Inone embodiment, the biotin analog is detected using an antibody. Thebiotin analog may be detected using a detection system such as but notlimited to fluorescent detection system, a luminescent detection system,a photographic film detection system, an enzyme detection system, anelectron spin resonance detection system, a scanning tunnelingmicroscopy (STM) detection system, an optical detection system and anuclear magnetic resonance (NMR) detection system.

In one embodiment, the method further comprises removing unbound biotinanalog prior to detecting bound biotin analog. The method may alsofurther comprise identifying a biotin ligase mutant having specificityfor a biotin analog and biotin. In a related embodiment, the biotinligase mutant having specificity for a biotin analog and biotin isidentified by contacting biotin with an acceptor peptide in the presenceof a candidate molecule, and detecting biotin that is bound to theacceptor peptide, wherein the presence of biotin bound to an acceptorpeptide indicates that the candidate molecule is a biotin ligase mutanthaving specificity for a biotin analog and biotin.

The method may also further comprise isolating the candidate moleculethat is a biotin ligase mutant having specificity for a biotin analog orthe biotin ligase mutant having specificity for a biotin analog andbiotin.

In another aspect, the invention provides a method for identifying abiotin analog having specificity for a biotin ligase mutant comprisingcombining an acceptor peptide with a labeled biotin in the presence of abiotin ligase mutant and determining a control level of biotinincorporation, combining an acceptor peptide with a labeled biotin and acandidate biotin analog molecule in the presence of a biotin ligasemutant and determining a test level of biotin incorporation, andcomparing the control and test levels of biotin incorporation, wherein atest level that is less than a control level is indicative of a biotinanalog having specificity for a biotin ligase mutant. Variousembodiments relating to the biotin ligase mutant, the biotin analog andthe acceptor peptide are recited above.

These and other objects of the invention will be described in furtherdetail in connection with the detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows biotinylation of the lysine side chain of the consensuspeptide sequence (SEQ ID NO: 5) of biotin ligase (BirA). (Chapman-Smithet al. J. Nutr., 129:477S–484S, 1999).

FIG. 1B shows the structures of biotin as well as various biotinanalogs. NBD-GABA (7-nitrobenz-2-oxa-1,3-diazole γ-aminobutyric acid) isa fluorophore with a similar size and shape to biotin. Biotin isostere(labeled as ketone) has a bio-orthogonal ketone functionality that canbe chemoselectively modified with hydrazine- and alkoxyamine-derivatizedprobes as shown in FIG. 2. (Cornish et al. J. Am. Chem. Soc. 118,8150–8151, 1996; Mahal et al. Science 276, 1125–1128, 1997.) Coumarinand fluorescein are directly detectable biotin analogs.

FIG. 2 shows the labeling of biotin analogs with labels. Biotin analogsthat introduce unique chemical handles for subsequent modification by arange of probes in the live cell context are shown. “F” represents anyfluorophore. The ketone biotin analog can be selectively conjugated tohydrazide, hydroxylamino, and thiosemicarbazide groups underphysiological conditions. The azide biotin analog can be selectivelycoupled to phosphines via the modified Staudinger reaction. (Saxon andBertozzi, Science 287:2007–2010, 2000.) The tetrathiol biotin analog canform a stable adduct with the fluorescein-arsenic derivative (FlAsH)shown. The reaction of azide with a fluorogenic biotin analog (e.g.,non-fluorescent coumarin phosphine) results in a detectable compound(e.g., fluorescent coumarin).

FIG. 3A shows a phage display scheme to select for desired biotin ligasemutants from a library. Wild type biotin ligase has already beensuccessfully displayed on phage and enriched in model selections by Neriet al. (Heinis et al. Protein Engineering 14:1043–1052, 2001.)

FIG. 3B shows the results of biotinylation activity assays for wild typebiotin ligase in soluble or phage displayed form, and either in thepresence or absence of ATP.

FIG. 4 shows a synthesis pathway for the ketone biotin analog.

FIG. 5 shows a synthesis pathway for the N-acyl azide and NBD-GABAbiotin analogs.

FIG. 6A shows expression of wild type biotin ligase and biotin ligasemutants.

FIG. 6B shows the results of biotinylation activity assays for variousbiotin ligase mutants. The biotin ligase mutants harboring amino acidsubstitutions of T90G, G115A or T90V have affinity for biotin comparableto wild type biotin ligase.

FIG. 7 shows the alignment of the amino acid (SEQ ID NO:1) andnucleotide (SEQ ID NO:2) sequence of wild type biotin ligase.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO: 1 is the amino acid sequence of wild type biotin ligase.

SEQ ID NO: 2 is the nucleotide sequence of wild type biotin ligase.

SEQ ID NO: 3 is a consensus amino acid sequence of an acceptor peptide.

SEQ ID NO: 4 is the amino acid sequence of a 13 amino acid acceptorpeptide.

SEQ ID NO: 5 is the amino acid sequence of an acceptor peptide(AviTag™).

SEQ ID NO: 6 is the amino acid sequence of a biotin ligase mutant havinga T90G amino acid substitution.

SEQ ID NO: 7 is the amino acid sequence of a biotin ligase mutant havingT90G and N91S amino acid substitutions.

SEQ ID NO: 8 is the amino acid sequence of a biotin ligase mutant havingpossible amino acid substitutions at amino acid positions 83, 89, 90,91, 92, 107, 112, 115, 116, 117, 118, 123, 132, 134, 142, 186, 188, 189,190, 204, 206, 207, or 235.

SEQ ID NO: 9 is the amino acid sequence of a biotin ligase mutant havingT90G, T90A, or T90V amino acid substitutions.

SEQ ID NO: 10 is the amino acid sequence of a biotin ligase mutanthaving T90G, T90A, or T90V and N91S, N91G, N91A, or N91L amino acidsubstitutions.

SEQ ID NO: 11 is the amino acid sequence of a biotin ligase mutanthaving T90G and N91G amino acid substitutions.

SEQ ID NO: 12 is the amino acid sequence of a biotin ligase mutanthaving T90A and N91A amino acid substitutions.

SEQ ID NO: 13 is the amino acid sequence of a biotin ligase mutanthaving T90A and N91L amino acid substitutions.

SEQ ID NO: 14 is the amino acid sequence of a biotin ligase mutanthaving C107G amino acid substitution.

SEQ ID NO: 15 is the amino acid sequence of a biotin ligase mutanthaving Q112M amino acid substitution.

SEQ ID NO: 16 is the amino acid sequence of a biotin ligase mutanthaving G115A amino acid substitution.

SEQ ID NO: 17 is the amino acid sequence of a biotin ligase mutanthaving Y132G amino acid substitution.

SEQ ID NO: 18 is the amino acid sequence of a biotin ligase mutanthaving Y132A amino acid substitution.

SEQ ID NO: 19 is the amino acid sequence of a biotin ligase mutanthaving S134G amino acid substitution.

SEQ ID NO: 20 is the amino acid sequence of a biotin ligase mutanthaving V189G amino acid substitution.

SEQ ID NO: 21 is the amino acid sequence of a biotin ligase mutanthaving I207S amino acid substitution.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to protein labeling in vivo and in vitro. Priorattempts to label specific proteins have been frustrated by a lack ofreagents with sufficient specificity. The invention aims to overcomethis lack of specificity through the use of biotin ligase mutants andbiotin analogs that are recognized by such mutants.

The invention therefore provides, inter alia, methods for labelingproteins in vitro or in vivo. The method generally involves contacting abiotin analog with a fusion protein in the presence of a biotin ligasemutant, and allowing sufficient time for conjugation of the biotinanalog to the fusion protein. Times and reaction conditions suitable forbiotin ligase mutant activity will generally be comparable to those forwild type biotin ligase which are known in the art. (See for exampleExamples herein and Avidity technical literature.)

The various components of this reaction will be described in greaterdetail herein. Briefly, the fusion protein is a fusion of the targetprotein (i.e., the protein which is to be labeled) and an acceptorpeptide (i.e., the peptide sequence that acts as a substrate for thebiotin ligase mutant). If the method is performed in vivo, the nucleicacid sequence encoding the fusion protein will be introduced into thecell and transcription and translation allowed to occur. If the methodis performed in vitro, the fusion protein will simply be added to thereaction mixture.

As used herein, protein labeling in vitro means labeling of a protein ina cell free environment. As an example, such a protein can be combinedwith a biotin ligase mutant and a biotin analog under appropriateconditions and thereby labeled, in for example a test tube or a well ofa multiwell plate.

As used herein, protein labeling in vivo means labeling of a protein inthe context of a cell. The method can be used to label proteins that areintracellular proteins or cell surface proteins. The cell may be presentin a subject (e.g., an insect such as Drosophila, a rodent such as amouse, a human, and the like) or it may be present in culture.

The biotin ligase mutant may also be expressed by the cell in someinstances. In other instances, however, the biotin ligase mutant maysimply be added to the reaction mixture (if in vitro) or to the cell (ifthe target protein is a cell surface protein and the acceptor peptide islocated on the extracellular domain of the target protein).

According to the method, the biotin ligase mutant conjugates the biotinanalog to the acceptor peptide that is fused (either at the nucleic acidlevel or post-translationally) to the target protein. The method isindependent of the protein type and thus any protein can be labeled inthis manner. The product of this labeling reaction may or may not bedirectly detectable however depending upon the nature of the biotinanalog, as described herein. Accordingly, it may be necessary to reactthe conjugated biotin analog with a detectable label. If the method isperformed in vivo, the detectable label is preferably one capable ofdiffusion into a cell. If the method is used to label a cell surfaceprotein, then preferably the biotin analog is labeled with a membraneimpermeant label in order to reduce entry and accumulation of the labelintracellularly. The biotin analog may be labeled prior to or afterconjugation to the fusion protein.

Labeling of proteins allows one to track the movement and activity ofsuch proteins. It also allows cells expressing such proteins to betracked and imaged, as the case may be. The methods can be used in cellsfrom virtually any organism including insect, yeast, frog, worm, fish,rodent, human and the like.

The method can be used to label virtually any protein. Examples includebut are not limited to signal transduction proteins (e.g., cell surfacereceptors, kinases, adapter proteins), nuclear proteins (transcriptionfactors, histones), mitochondrial proteins (cytochromes, transcriptionfactors) and hormone receptors.

Biotin ligase (BirA) is an 321 amino acid, 33.5 kD enzyme derived fromE. coli that catalyzes the context-specific conjugation of biotin to alysine ε-amine in biotin retention and biosynthesis pathways, as shownin FIG. 1A. This reaction is ATP-dependent. As used herein, wild typebiotin ligase refers to a naturally occurring bacterial biotin ligasehaving wild type biotinylation activity. SEQ ID NO: 1 represents theamino acid sequence of wild type biotin ligase (GenBank Accession No.M10123). SEQ ID NO: 2 represents the nucleotide sequence of wild typebiotin ligase (GenBank Accession No. M10123).

Biotin ligase is also known as biotin protein ligase, biotin operonrepressor protein, BirA, biotin holoenzyme synthetase andbiotin-[acetyl-CoA carboxylase] synthetase.

The reaction between biotin ligase and its substrate (discussed below)is referred to as orthogonal. This means that neither the ligase nor itssubstrate react with any other enzyme or molecule when present either intheir native environment (i.e., a bacterial cell) or more importantlyfor the purposes of the invention in a non-native environment (e.g., amammalian cell). Accordingly, the invention takes advantage of the highdegree of specificity which has evolved between biotin ligase and itssubstrate.

The only known natural substrate in bacteria of wild type biotin ligaseis lysine 122 of the biotin carboxyl carrier protein (BCCP).Chapman-Smith et al. J. Nutr. 129:477S–484S, 1999.) A 13–15 amino acidminimal substrate sequence encompassing lysine 122 has been identifiedas the minimal peptide recognition sequence for biotin ligase. As usedherein, an “acceptor peptide” is a protein or peptide having an aminoacid sequence that is a substrate for a biotin ligase mutant (i.e., abiotin ligase mutant recognizes and is capable of conjugating a biotinanalog or biotin to the peptide). The acceptor peptide may have an aminoacid sequence of Leu Xaa₁ Xaa₂ Ile Xaa₃ Xaa₄ Xaa₅ Xaa₆ Lys Xaa₇ Xaa₈Xaa₉ Xaa₁₀ (SEQ. ID NO:3), where Xaa₁ is any amino acid, Xaa₂ is anyamino acid other than large hydrophobic amino acids (such as Leu, Val,Ile, Trp, Phe, Tyr); Xaa₃ is Phe or Leu, Xaa4 is Glu or Asp; Xaa₅ isAla, Gly, Ser, or Thr; Xaa₆ is Gln or Met; Xaa₇ is Ile, Met, or Val;Xaa₈ is Glu, Leu, Val, Tyr, or Ile; Xaa₉ is Trp, Tyr, Val, Phe, Leu, orIle; and Xaa₁₀ is preferably Arg or His but may be any amino acid otherthan acidic amino acids such as Asp or Glu. Acceptor peptides are knownin the art and examples are described in U.S. Pat. Nos. 5,723,584;5,874,239 and 5,932,433, the entire contents of which are hereinincorporated by reference. In important embodiments, the acceptorpeptide comprises the amino acid sequence of LNDIFEAQKIEWH (SEQ ID NO:4). In another embodiment, the acceptor peptide comprises an amino acidsequence of GLNDIFEAQKIEWHE (SEQ ID NO: 5). Acceptor peptides can besynthesized using standard peptide synthesis techniques. They are alsocommercially available under the trade name AviTag™ from Avidity(Boulder, Colo.).

The acceptor peptide is used in the methods of the invention to tagtarget proteins that are to be labeled by biotin ligase mutants. Theacceptor peptide and target protein may be fused to each other either atthe nucleic acid or amino acid level. Recombinant DNA technology forgenerating fusion nucleic acids that encode both the target protein andthe acceptor peptide are known in the art. Additionally, the acceptorpeptide may be fused to the target protein post-translationally. Suchlinkages may include cleavable linkers or bonds which can be cleavedonce the desired labeling is achieved. Such bonds may be cleaved byexposure to a particular pH, or energy of a certain wavelength, and thelike. Cleavable linkers are known in the art. Examples includethiol-cleavable cross-linker 3,3′-dithiobis(succinimidyl proprionate),amine-cleavable linkers, and succinyl-glycine spontaneously cleavablelinkers.

The acceptor peptide can be fused to the target protein at any position.In some instances, it is preferred that the fusion not interfere withthe activity of the target protein, accordingly, the acceptor peptide isfused to the protein at positions that do not interfere with theactivity of the protein. Generally, the acceptor peptides can be C- orN-terminally fused to the target proteins. In still other instances, itis possible that the acceptor peptide is fused to the target protein atan internal position (e.g., a flexible internal loop). These proteinsare then susceptible to specific tagging by biotin ligase and biotinligase mutants in vivo and in vitro. This specificity is possiblebecause neither biotin ligase nor the acceptor peptide react with anyother enzymes or peptides in a cell.

Thus, the invention is directed in part to generating biotin ligasemutants that recognize biotin analogs and conjugate such analogs to theacceptor peptide. Biotin ligase mutants can be generated in any numberof ways, including phage display technology, described in greater detailherein.

The labeling methods of the invention rely on the activity of biotinligase mutants that recognize and conjugate biotin analogs onto fusionproteins via the acceptor peptide. The invention provides biotin ligasemutants that recognize biotin analogs, and in some instances, biotinitself. As used herein, a biotin ligase mutant is a variant of biotinligase that is enzymatically active towards a biotin analog (such asthose described herein). As used herein, “enzymatically active” meansthat the mutant is able to recognize and conjugate a biotin analog tothe acceptor peptide.

The biotin ligase mutant can have various mutations, including addition,deletion or substitution of one or more amino acids. Preferably, themutation will be present in the biotin interaction and activationregion, spanning amino acids 83–235. Generally, these mutants willpossess one or more amino acid substitutions relative to the wild typebiotin ligase amino acid sequence (SEQ ID NO:1). In most instances, thebiotin ligase mutants do not comprise an amino acid substitution (orother form of mutation) at position 183 (which is the putative catalyticresidue) or residues near the peptide binding site and/or the ATPbinding site (amino acids 1–26).

Some mutants were developed based on an analysis of the biotin bindingsite of wild type biotin ligase, particularly in the presence of biotin.Residues that appear important in the interaction with biotin include89–91, 112, 115–118, 123, 186, 190, 204 and 206. Residues that influencebiotin affinity include 83, 107, 115, 118, 142, 189, 207 and 235. Bothtypes of residues are included in the biotin interaction and activationdomain. In some important embodiments of the invention, mutants compriseamino acid substitutions at one or more of the following positions: T90,N91, C107, Q112, G115, R116, Y132, S134, L188, V189, I207. Specificexamples of biotin ligase mutants are proteins having at least one ofthe following amino acid substitutions: T90G, T90A, T90V, C107G, Q112M,G115A, Y132A, Y132G, S134G, V189G and I207S. The invention contemplatesthe use of biotin ligase mutants having an amino acid substitution atone or more of the afore-mentioned positions. Of particular importanceare biotin ligase mutants that harbor amino acid substitutions atpositions T90 and N91. Examples include but are not limited toT90G/N91S, T90G/N91G, T90A/N91A, T90A/N91 L and T90V/N91L.

The biotin ligase mutant may retain some level of activity for biotin.Its binding affinity for biotin may be similar to that of wild typebiotin ligase. Preferably, the mutant has higher binding affinity for abiotin analog than it does for biotin. Consequently, biotin conjugationto an acceptor peptide would be lower in the presence of a biotinanalog. In still other embodiments, the biotin ligase mutant has nobinding affinity for biotin.

Biotin incorporation can be measured using ³H-biotin and measuringincorporation of radioisotope in the peptide. Conjugation of the biotinanalog to an acceptor peptide can be assayed based on inhibition ofbiotin incorporation. In this latter assay, incorporation of a biotinanalog is indicated by a reduced amount of incorporated radioactivitysince the biotin analog is competed with biotin for conjugation to theacceptor peptide.

The skilled artisan will realize that conservative amino acidsubstitutions may be made in biotin ligase mutants to providefunctionally equivalent variants, i.e., the variants retain thefunctional capabilities of the particular biotin ligase mutant. As usedherein, a “conservative amino acid substitution” refers to an amino acidsubstitution which does not alter the relative charge or sizecharacteristics of the protein in which the amino acid substitution ismade. Variants can be prepared according to methods for alteringpolypeptide sequence known to one of ordinary skill in the art such asare found in references which compile such methods, e.g. MolecularCloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, orCurrent Protocols in Molecular Biology, F. M. Ausubel, et al., eds.,John Wiley & Sons, Inc., New York. Conservative substitutions of aminoacids include substitutions made amongst amino acids within thefollowing groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G;(e) S, T; (f) Q, N; and (g) E, D.

Conservative amino-acid substitutions in the amino acid sequence ofbiotin ligase mutants to produce functionally equivalent variantstypically are made by alteration of a nucleic acid encoding the mutant.Such substitutions can be made by a variety of methods known to one ofordinary skill in the art. For example, amino acid substitutions may bemade by PCR-directed mutation, site-directed mutagenesis according tothe method of Kunkel (Kunkel, PNAS 82: 488–492, 1985), or by chemicalsynthesis of a nucleic acid molecule encoding a biotin ligase mutant.

Similarly, biotin ligase mutants can be made using standard molecularbiology techniques known to those of ordinary skill in the art. Forexample, the mutants may be formed by transcription and translation froma nucleic acid sequence encoding the mutant. Such nucleic acid sequencescan be made based on the teaching of wild type biotin ligase sequenceand the position and type of amino acid substitution.

The invention further provides methods for screening candidate moleculesfor activity as a biotin ligase mutant. These screening methods can alsobe combined with methods for generating candidates. One example is aphage display library in which the candidates can be generated and alsotested for their ability to conjugate a biotin analog to an acceptorpeptide. This is illustrated in FIG. 3 which demonstrates the use ofphage having the acceptor peptide present on their coat. Phage thatdisplay “active” biotin ligase mutants (i.e., mutants that are able toconjugate a biotin analog (in this case a fluorophore bearing biotinanalog) to the acceptor peptide are selected for (using an antibody tothe fluorophore). The phage can then optionally be further manipulatedto generate derivatives of the active mutant. Phage display librarytechnology is known in the art and has been described extensively. (Seefor example Benhar, Biotechnol Adv. 2001 Feb. 1;19(1):1–33;Anthony-Cahill et al. Curr Pharm Biotechnol. 2002 December;3(4):299–315,among others.)

The labeling methods of the invention further rely on biotin analogsthat are recognized and conjugated to acceptor peptides by biotin ligasemutants. As used herein, a biotin analog is a molecule that isstructurally similar to biotin. (See for example the structuralsimilarity between ketone biotin analog, azide biotin analog and biotin,as shown in FIG. 1B.) Biotin analogs may share one particular structuralfeature in common with biotin such as for example an aliphaticcarboxylic tail, a two-ring structure, and the like. A biotin analog maybe synthesized from biotin, but is not so limited. Examples of biotinanalogs of this latter class include biotin methyl ester, desthiobiotin,2′-iminobiotin, and diaminobiotin. The biotin ligase mutants must becapable of recognizing and conjugating biotin analogs to acceptorpeptides, in a manner similar to that in which wild type biotin ligaserecognizes and conjugates biotin to the acceptor peptide.

The biotin analog binds to a biotin ligase mutant in the interaction andactivation domain. Preferably it binds with an affinity comparable tothe binding affinity of wild type biotin ligase to biotin. However,biotin analogs that bind with lower affinities are still usefulaccording to the invention. In some important embodiments, the biotinanalog is not recognized by wild type biotin ligase derived from eitherE. coli or from other cell types (e.g., the cell in which the labelingreaction is proceeding).

One category of biotin analogs are molecules having an aliphaticcarboxylic acid tail. Examples are shown in FIG. 1B. These include butare not limited to ketone biotin analog, N-ketone biotin analog (e.g.,biotin isostere), N-alkyne biotin analog, azide biotin analog, N-acylazide biotin analog, N-azide biotin analog, coumarin, fluorescein, NBDand 1,2-diamine biotin analog.

Biotin analogs may comprise substitutions (e.g., alkylation) at thetrans-ureido nitrogen of biotin. Examples include N-ketone biotinanalog, N-alkyne biotin, N-azide and N-acyl azide, all of which areillustrated in FIG. 1B.

Some biotin analogs are not themselves directly detectable, while othersare. In the case of the former type, the biotin analog undergoesreaction with another moiety (either before or after conjugation to theacceptor peptide). The subsequent modification of this former type ofbiotin analog is referred to as a bio-orthogonal ligation reaction andit is used to couple (i.e., label) these biotin analogs to detectablelabels such as fluorophores. The resulting moiety may be a hydrazide,phosphine, or azide, but is not so limited. Examples of this former typeof biotin analog include ketone biotin analogs, azide biotin analogs,N-acyl azide biotin analogs, N-azide biotin analogs, and tetrathiolbiotin analogs, among others. The structures of these biotin analogs areillustrated in FIG. 1B.

FIG. 4 illustrates the synthesis of a ketone biotin analog. FIG. 5illustrates the synthesis of azide and NBD biotin analogs. Thesesynthesis pathways are exemplary and other synthesis protocols can beused to generate these biotin analogs.

Accordingly, biotin analogs that are not themselves directly detectablemust be reacted with a detectable moiety. Each biotin analog in thiscategory will undergo a specific reaction dependent upon its functionalgroups and that of its reaction partner. Some of these reactions areshown in FIG. 2. The reaction partners in FIG. 2 arefluorophore-bearing, however it is to be understood that the reactionpartner may comprise any detectable moiety and is not solely limited tofluorophores. For example, a ketone biotin analog may be reacted with ahydrazine to form a hydrazone. Ketone-hydrazide ligation is fairly rapidand works with high specificity on cell surfaces. (Mahal et al. Science276:1125–1128, 1997.)

In another example, azides may be reacted with phosphines in aStaudinger reaction. Azides and aryl phosphines generally have nocellular counterparts. As a result, the reaction is quite specific.Azide variants with improved stability against hydrolysis in water at pH6–8 are also useful in the methods of the invention. The alkyne/azide[3+2] cycloaddition chemistry, based on Click chemistry (Wang et al. J.Am. Chem. Soc. 125:11164–11165, 2003), is also specific, in part becausethe two reactive partners do not have cellular counterparts (i.e., thetwo functional groups are non-naturally occurring).

As stated above, other biotin analogs may be themselves directlydetectable. Examples of such biotin analogs include but are not limitedto NBD-GABA, coumarin, fluorescein, Texas Red® (sulforhodamine 101),rhodamine, rosamine, Alexa™ dyes, resorufin, oregon green, tetramethylrhodamine (TMR), carboxy tetramethyl-rhodamine (TAMRA),Carboxy-X-rhodamine (ROX), BODIPY dyes, and derivatives thereof. Severalof these dyes are known in the art and are commercially available (e.g.,from Molecular Probes). Several of these molecules are examples ofbiotin analogs that are not derived from biotin per se. Nonetheless theyshare structural similarity with biotin, making them suitable biotinanalogs for use in the methods of the invention.

The biotin analogs can also be fluorogenic. As used herein, afluorogenic compound is one that is not detectable (e.g., fluorescent)by itself, but when conjugated to another moiety becomes fluorescent. Anexample of this is non-fluorescent coumarin phosphine which reacts withazides to produce fluorescent coumarin. Another example of a fluorogenicbiotin analog is the diamine biotin analog shown in FIG. 1B. This analogcan undergo a condensation with diaminobenzaldehyde to form afluorescent adduct. (Leandri et al. Gazz. Chim. Ital. 769–839, 1955.)Fluorogenic biotin analogs are especially useful to keeping backgroundto a minimum (e.g., cellular imaging applications).

The invention therefore provides methods for using the afore-mentionedbiotin analogs, as well as compositions comprising some of theseanalogs. For example, the invention provides compositions comprising theNBD-GABA analog, as well as analogs alkyated at the trans-ureidonitrogen group of biotin (e.g., N-ketone biotin analog, ketone biotinanalog, N-alkyne biotin analog, N-acyl azide biotin analog and N-azidebiotin analog; see FIG. 1B).

As stated above, the biotin analogs can be conjugated to detectablelabels. A “detectable label” as used herein is a molecule or compoundthat can be detected by a variety of methods including fluorescence,electrical conductivity, radioactivity, size, and the like. The labelmay be of a chemical (e.g., carbohydrate, lipid, etc.), peptide ornucleic acid nature although it is not so limited. The label may bedirectly or indirectly detectable. The label can be detected directlyfor example by its ability to emit and/or absorb light of a particularwavelength. A label can be detected indirectly by its ability to bind,recruit and, in some cases, cleave (or be cleaved by) another compound,thereby emitting or absorbing energy. An example of indirect detectionis the use of an enzyme label which cleaves a substrate into visibleproducts.

The type of label used will depend on a variety of factors, such as butnot limited to the nature of the protein ultimately being labeled. Thelabel should be sterically and chemically compatible with the biotinanalog, the acceptor peptide and the target protein. In most instances,the label should not interfere with the activity of the target protein.

Generally, the label can be selected from the group consisting of afluorescent molecule, a chemiluminescent molecule (e.g.,chemiluminescent substrates), a phosphorescent molecule, a radioisotope,an enzyme, an enzyme substrate, an affinity molecule, a ligand, anantigen, a hapten, an antibody, an antibody fragment, a chromogenicsubstrate, a contrast agent, an MRI contrast agent, a PET label, aphosphorescent label, and the like.

Specific examples of labels include radioactive isotopes such as ³²P or³H; haptens such as digoxigenin and dintrophenyl; affinity tags such asa FLAG tag, an HA tag, a histidine tag, a GST tag; enzyme tags such asalkaline phosphatase, horseradish peroxidase, beta-galactosidase, etc.Other labels include fluorophores such as fluorescein isothiocyanate(“FITC”), Texas Red®, tetramethylrhodamine isothiocyanate (“TRITC”), 4,4-difluoro-4-bora-3a, and 4a-diaza-s-indacene (“BODIPY”), Cy-3, Cy-5,Cy-7, Cy-Chrome™, R-phycoerythrin (R-PE), PerCP, allophycocyanin (APC),PharRed™, Mauna Blue, Alexa™ 350 and other Alexa™ dyes, and CascadeBlue®.

The labels can also be antibodies or antibody fragments or theircorresponding antigen, epitope or hapten binding partners. Detection ofsuch bound antibodies and proteins or peptides is accomplished bytechniques well known to those skilled in the art. Antibody/antigencomplexes which form in response to hapten conjugates are easilydetected by linking a label to the hapten or to antibodies whichrecognize the hapten and then observing the site of the label.Alternatively, the antibodies can be visualized using secondaryantibodies or fragments thereof that are specific for the primaryantibody used. Polyclonal and monoclonal antibodies may be used.Antibody fragments include Fab, F(ab)₂, Fd and antibody fragments whichinclude a CDR3 region. The conjugates can also be labeled using dualspecificity antibodies.

The label can be a contrast agent. Contrast agents are molecules thatare administered to a subject to enhance a particular imaging modalitysuch as but not limited to X-ray, ultrasound, and MRI. Examples ofcontrast agents for transesophageal echocardiography (TEE) andtranscranial Doppler sonography: Echovist((R))-300 ( (TCD)); for MRI:superparamagnetic vascular contrast agent (MION), gadolinium(III),Gd-DTPA-BMA, superparamagnetic iron oxide (SPIO) SH U 555 A, gadoxeticacid; for ultrasonographic (US) angiography: microbubble-based UScontrast agent (FS069); for computed tomography: iopamidol; for X-rayvenography: NC100150.

The label can be a positron emission tomography (PET) label such as 99 mtechnetium and 18FDG.

The label can also be an singlet oxygen radical generator including butnot limited to resorufin, malachite green, fluorescein, benzidine andits analogs including 2-aminobiphenyl, 4-aminobiphenyl,3,3′-diaminobenzidine, 3,3′-dichlorobenzidine, 3,3′-dimethoxybenzidine,and 3,3′-dimethylbenzidine. These molecules are useful in EM stainingand can also be used to induce localized toxicity.

The label can also be an analyte-binding group such as but not limitedto a metal chelator (e.g., a copper chelator). Examples of metalchelators include EDTA, EGTA, and molecules having pyridiniumsubstituents, imidazole substituents, and/or thiol substituents. Theselabels can be used to analyze local environment of the target protein(e.g., Ca²⁺ concentration).

The label can also be a heavy atom carrier. Such labels would beparticularly useful for X-ray crystallographic study of the targetprotein. Heavy atoms used in X-ray crystallography include but are notlimited to Au, Pt and Hg. An example of a heavy atom carrier is iodine.

The label may also be a photoactivatable cross-linker. A photoactivablecross linker is a cross linker that becomes reactive following exposureto radiation (e.g., a ultraviolet radiation, visible light, etc.).Examples include benzophenones, aziridines, a photoprobe analog ofgeranylgeranyl diphosphate (2-diazo-3,3,3-trifluoropropionyloxy-farnesyldiphosphate or DATFP-FPP) (Quellhorst et al. J Biol Chem. 2001 Nov.2;276(44):40727–33), a DNA analogue5-[N-(p-azidobenzoyl)-3-aminoallyl]-dUTP (N(3)RdUTP),sulfosuccinimidyl-2(7-azido-4-methylcoumarin-3-acetamido)-ethyl-1,3′-dithiopropionate (SAED) and1-[N-(2-hydroxy-5-azidobenzoyl)-2-aminoethyl]-4-(N-hydroxysuccinimidyl)-succinate.

The label may also be a photoswitch label. A photoswitch label is amolecule that undergoes a conformational change in response toradiation. For example, the molecule may change its conformation fromcis to trans and back again in response to radiation. The wavelengthrequired to induce the conformational switch will depend upon theparticular photoswitch label. Examples of photoswitch labels includeazobenzene, 3-nitro-2-naphthalenemethanol. Examples of photoswitches arealso described in van Delden et al. Chemistry. 2004 Jan. 5;10(1):61–70;van Delden et al. Chemistry. 2003 Jun. 16;9(12):2845–53; Zhang et al.Bioconjug Chem. 2003 July–August;14(4):824–9; Irie et al. Nature. 2002Dec. 19–26;420(6917):759–60; as well as many others.

The label may also be a photolabile protecting group. Examples ofphotolabile protecting group include a nitrobenzyl group, a dimethoxynitrobenzyl group, nitroveratryloxycarbonyl (NVOC),2-(dimethylamino)-5-nitrophenyl (DANP), Bis(o-nitrophenyl)ethanediol,brominated hydroxyquinoline, and coumarin-4-ylmethyl derivative.Photolabile protecting groups are useful for photocaging reactivefunctional groups.

The label may comprise non-naturally occurring amino acids. Examples ofnon-naturally occurring amino acids include for glutamine (Glu) orglutamic acid residues: α-aminoadipate molecules; for tyrosine (Tyr)residues: phenylalanine (Phe), 4-carboxymethyl-Phe, pentafluorophenylalanine (PfPhe), 4-carboxymethyl-L-phenylalanine (cmPhe),4-carboxydifluoromethyl-L-phenylalanine (F₂cmPhe),4-phosphonomethyl-phenylalanine (Pmp),(difluorophosphonomethyl)phenylalanine (F₂Pmp), O-malonyl-L-tyrosine(malTyr or OMT), and fluoro-O-malonyltyrosine (FOMT); for prolineresidues: 2-azetidinecarboxylic acid or pipecolic acid (which have6-membered, and 4-membered ring structures respectively);1-aminocyclohexylcarboxylic acid (Ac₆c);3-(2-hydroxynaphtalen-1-yl)-propyl; S-ethylisothiourea;2-NH₂-thiazoline; 2-NH₂-thiazole; asparagine residues substituted with3-indolyl-propyl at the C terminal carboxyl group. Modifications ofcysteines, histidines, lysines, arginines, tyrosines, glutamines,asparagines, prolines, and carboxyl groups are known in the art and aredescribed in U.S. Pat. No. 6,037,134. These types of labels can be usedto study enzyme structure and function.

The label may be an enzyme or an enzyme substrate. Examples of theseinclude (enzyme (substrate)): Alkaline Phosphatase (4-Methylumbelliferylphosphate Disodium salt; 3-Phenylumbelliferyl phosphate Hemipyridinesalt); Aminopeptidase (L-Alanine-4-methyl-7-coumarinylamidetrifluoroacetate; Z-L-arginine-4-methyl-7-coumarinylamide hydrochloride;Z-glycyl-L-proline-4-methyl-7-coumarinylamide); Aminopeptidase B(L-Leucine-4-methyl-7-coumarinylamide hydrochloride); Aminopeptidase M(L-Phenylalanine 4-methyl-7-coumarinylamide trifluoroacetate); Butyrateesterase (4-Methylumbelliferyl butyrate); Cellulase(2-Chloro-4-nitrophenyl-beta-D-cellobioside); Cholinesterase(7-Acetoxy-1-methylquinolinium iodide; Resorufin butyrate);alpha-Chymotrypsin, (Glutaryl-L-phenylalanine4-methyl-7-coumarinylamide);N-(N-Glutaryl-L-phenylalanyl)-2-aminoacridone;N-(N-Succinyl-L-phenylalanyl)-2-aminoacridone); Cytochrome P450 2B6(7-Ethoxycoumarin); Cytosolic Aldehyde Dehydrogenase (Esterase Activity)(Resorufin acetate); Dealkylase (O⁷-Pentylresorufin); Dopaminebeta-hydroxylase (Tyramine); Esterase (8-Acetoxypyrene-1,3,6-trisulfonicacid Trisodium salt; 3-(2 Benzoxazolyl)umbelliferyl acetate;8-Butyryloxypyrene-1,3,6-trisulfonicacid Trisodium salt;2′,7′-Dichlorofluorescin diacetate; Fluorescein dibutyrate; Fluoresceindilaurate; 4-Methylumbelliferyl acetate; 4-Methylumbelliferyl butyrate;8-Octanoyloxypyrene-1,3,6-trisulfonic acid Trisodium salt;8-Oleoyloxypyrene-1,3,6-trisulfonic acid Trisodium salt; Resorufinacetate); Factor X Activated (Xa) (4-Methylumbelliferyl4-guanidinobenzoate hydrochloride Monohydrate); Fucosidase, alpha-L-(4-Methylumbelliferyl-alpha-L-fucopyranoside); Galactosidase,alpha-(4-Methylumbelliferyl-alpha-D galactopyranoside); Galactosidase,beta- (6,8-Difluoro-4-methylumbelliferyl-beta-D-galactopyranoside;Fluorescein di(beta-D-galactopyranoside);4-Methylumbelliferyl-alpha-D-galactopyranoside;4-Methylumbelliferyl-beta-D-lactoside:Resorufin-beta-D-galactopyranoside;4-(Trifluoromethyl)umbelliferyl-beta-D-galactopyranoside;2-Chloro-4-nitrophenyl-beta-D-lactoside); Glucosaminidase,N-acetyl-beta- (4-Methylumbelliferyl-N-acetyl-beta-D-glucosaminideDihydrate); Glucosidase,alpha-(4-Methylumbelliferyl-alpha-D-glucopyranoside); Glucosidase, beta-(2-Chloro-4-nitrophenyl-beta-D-glucopyranoside;6,8-Difluoro-4-methylumbelliferyl-beta-D-glucopyranoside;4-Methylumbelliferyl-beta-D-glucopyranoside;Resorufin-beta-D-glucopyranoside;4-(Trifluoromethyl)umbelliferyl-beta-D-glucopyranoside); Glucuronidase,beta-(6,8-Difluoro-4-methylumbelliferyl-beta-D-glucuronide Lithium salt;4-Methylumbelliferyl-beta-D-glucuronide Trihydrate); Leucineaminopeptidase( L-Leucine-4-methyl-7-coumarinylamide hydrochloride);Lipase (Fluorescein dibutyrate; Fluorescein dilaurate;4-Methylumbelliferyl butyrate; 4-Methylumbelliferyl enanthate;4-Methylumbelliferyl oleate; 4-Methylumbelliferyl palmitate; Resorufinbutyrate); Lysozyme(4-Methylumbelliferyl-N,N′,N′-triacetyl-beta-chitotrioside);Mannosidase, alpha- (4-Methylumbelliferyl -alpha-D-mannopyranoside);Monoamine oxidase (Tyramine); Monooxygenase (7-Ethoxycoumarin);Neuraminidase (4-Methylumbelliferyl-N-acetyl-alpha-D-neuraminic acidSodium salt Dihydrate); Papain (Z-L-arginine-4-methyl-7-coumarinylamidehydrochloride); Peroxidase (Dihydrorhodamine 123); Phosphodiesterase(1-Naphthyl 4-phenylazophenyl phosphate; 2-Naphthyl 4-phenylazophenylphosphate); Prolyl endopeptidase(Z-glycyl-L-proline-4-methyl-7-coumarinylamide;Z-glycyl-L-proline-2-naphthylamide; Z-glycyl-L-proline-4-nitroanilide);Sulfatase (4-Methylumbelliferyl sulfate Potassium salt); Thrombin(4-Methylumbelliferyl 4-guanidinobenzoate hydrochloride Monohydrate);Trypsin (Z-L-arginine-4-methyl-7-coumarinylamide hydrochloride;4-Methylumbelliferyl 4-guanidinobenzoate hydrochloride Monohydrate);Tyramine dehydrogenase (Tyramine).

It is to be understood that many of the foregoing labels can also bebiotin analogs. That is, depending upon the particular biotin ligasemutant used, the various afore-mentioned labels may function as biotinanalogs. As such, these biotin analogs would be considered to bedirectly detectable biotin analogs. In some cases, they would notrequire further modification.

The labels can be attached to the biotin analogs either before or afterthe analog has been conjugated to the acceptor peptide, presuming thatthe label does not interfere with the activity of biotin ligase. Labelscan be attached to the biotin analogs by any mechanism known in the art.Some of these mechanisms are already described above for particularanalogs. Other examples of functional groups which are reactive withvarious labels include, but are not limited to, (functional group:reactive group of light emissive compound) activated ester:amines oranilines; acyl azide:amines or anilines; acyl halide:amines, anilines,alcohols or phenols; acyl nitrile:alcohols or phenols; aldehyde:aminesor anilines; alkyl halide:amines, anilines, alcohols, phenols or thiols;alkyl sulfonate:thiols, alcohols or phenols; anhydride:alcohols,phenols, amines or anilines; aryl halide:thiols; aziridine:thiols orthioethers; carboxylic acid:amines, anilines, alcohols or alkyl halides;diazoalkane:carboxylic acids; epoxide:thiols; haloacetamide:thiols;halotriazine:amines, anilines or phenols; hydrazine:aldehydes orketones; hydroxyamine:aldehydes or ketones; imido ester:amines oranilines; isocyanate:amines or anilines; and isothiocyanate:amines oranilines.

The labels are detected using a detection system. The nature of suchdetection systems will depend upon the nature of the detectable label.The detection system can be selected from any number of detectionsystems known in the art. These include a fluorescent detection system,a photographic film detection system, a chemiluminescent detectionsystem, an enzyme detection system, an atomic force microscopy (AFM)detection system, a scanning tunneling microscopy (STM) detectionsystem, an optical detection system, a nuclear magnetic resonance (NMR)detection system, a near field detection system, and a total internalreflection (TIR) detection system.

The invention provides in some instances biotin ligase mutants and/orbiotin analogs in an isolated form. As used herein, an isolated biotinligase mutant is a biotin ligase mutant that is separated from itsnative environment in sufficiently pure form so that it can bemanipulated or used for any one of the purposes of the invention. Thus,isolated means sufficiently pure to be used (i) to raise and/or isolateantibodies, (ii) as a reagent in an assay, or (iii) for sequencing, etc.

Isolated biotin analogs similarly are analogs that have beensubstantially separated from either their native environment (if itexists in nature) or their synthesis environment. Accordingly, thebiotin analogs are substantially separated from any or all reagentspresent in their synthesis reaction that would be toxic or otherwisedetrimental to the target protein, the acceptor peptide, the biotinligase mutant, or the labeling reaction. Isolated biotin analogs, forexample, include compositions that comprise less than 25% contamination,less than 20% contamination, less than 15% contamination, less than 10%contamination, less than 5% contamination, or less than 1% contamination(w/w).

The invention further provides nucleic acids coding for biotin ligasemutants. These nucleic acids therefore encode a biotin ligase mutanthaving an amino acid substitution at one or more of the followingresidues: 83, 89–91, 107, 112, 115–118, 123, 132, 134, 142, 186, 188,189, 190, 204, 206, 207 and 235. In some important embodiments, theamino acid substitution is selected from the group consisting of T90G,T90A, T90V, C107G, Q112M, G115A, Y132A, Y132G, S134G, V189G and I207S.Nucleic acids that encode mutants having substitutions at two or moreresidues, such as T90G/N91S, T90G/N91G, T90A/N91A, T90A/N91 L andT90V/N91L, are also embraced by the invention.

The nucleotide sequence of wild type biotin ligase mutant is provided asSEQ ID NO: 2. One of ordinary skill in the art will be able to determinethe codons corresponding to each of the amino acid residues recitedherein.

The invention also embraces degenerate nucleic acids that differ fromthe mutant nucleic acid sequences provided herein in codon sequence dueto degeneracy of the genetic code. For example, serine residues areencoded by the codons TCA, AGT, TCC, TCG, TCT and AGC. Each of the sixcodons is equivalent for the purposes of encoding a serine residue.Thus, it will be apparent to one of ordinary skill in the art that anyof the serine-encoding nucleotide triplets may be employed to direct theprotein synthesis apparatus, in vitro or in vivo, to incorporate aserine residue into an elongating mutant. Similarly, nucleotide sequencetriplets which encode other amino acid residues include, but are notlimited to: CCA, CCC, CCG and CCT (proline codons); CGA, CGC, CGG, CGT,AGA and AGG (arginine codons); ACA, ACC, ACG and ACT (threonine codons);AAC and AAT (asparagine codons); and ATA, ATC and ATT (isoleucinecodons). Other amino acid residues may be encoded similarly by multiplenucleotide sequences.

The invention also involves expression vectors coding for biotin ligasemutants and host cells containing those expression vectors. Virtuallyany cells, prokaryotic or eukaryotic, which can be transformed withheterologous DNA or RNA and which can be grown or maintained in culture,may be used in the practice of the invention. Examples include bacterialcells such as E. coli , mammalian cells such as mouse, hamster, pig,goat, primate, etc., and other eukaryotic cells such as Xenopus cells,Drosophila cells, Zebrafish cells, C. elegans cells, and the like. Theymay be of a wide variety of tissue types, including mast cells,fibroblasts, oocytes and lymphocytes, and they may be primary cells orcell lines. Specific examples include CHO cells and COS cells. Cell-freetranscription systems also may be used in lieu of cells.

As used herein, a “vector” may be any of a number of nucleic acids intowhich a desired sequence may be inserted by restriction and ligation fortransport between different genetic environments or for expression in ahost cell. Vectors are typically composed of DNA although RNA vectorsare also available. Vectors include, but are not limited to, plasmids,phagemids and virus genomes. A cloning vector is one which is able toreplicate in a host cell, and which is further characterized by one ormore endonuclease restriction sites at which the vector may be cut in adeterminable fashion and into which a desired DNA sequence may beligated such that the new recombinant vector retains its ability toreplicate in the host cell. In the case of plasmids, replication of thedesired sequence may occur many times as the plasmid increases in copynumber within the host bacterium or just a single time per host beforethe host reproduces by mitosis. In the case of phage, replication mayoccur actively during a lytic phase or passively during a lysogenicphase.

An expression vector is one into which a desired DNA sequence may beinserted by restriction and ligation such that it is operably joined toregulatory sequences and may be expressed as an RNA transcript. Vectorsmay further contain one or more marker sequences (i.e., reportersequences) suitable for use in the identification of cells which have orhave not been transformed or transfected with the vector. Markersinclude, for example, genes encoding proteins which increase or decreaseeither resistance or sensitivity to antibiotics or other compounds,genes which encode enzymes whose activities are detectable by standardassays known in the art (e.g., beta-galactosidase or alkalinephosphatase), and genes which visibly affect the phenotype oftransformed or transfected cells, hosts, colonies or plaques. Preferredvectors are those capable of autonomous replication and expression ofthe structural gene products present in the DNA segments to which theyare operably joined.

As used herein, a marker or coding sequence and regulatory sequences aresaid to be “operably” joined when they are covalently linked in such away as to place the expression or transcription of the coding sequenceunder the influence or control of the regulatory sequences. If it isdesired that the coding sequences be translated into a functionalprotein, two DNA sequences are said to be operably joined if inductionof a promoter in the 5′ regulatory sequences results in thetranscription of the coding sequence and if the nature of the linkagebetween the two DNA sequences does not (1) result in the introduction ofa frame-shift mutation, (2) interfere with the ability of the promoterregion to direct the transcription of the coding sequences, or (3)interfere with the ability of the corresponding RNA transcript to betranslated into a protein. Thus, a promoter region would be operablyjoined to a coding sequence if the promoter region were capable ofeffecting transcription of that DNA sequence such that the resultingtranscript might be translated into the desired protein or polypeptide.

The precise nature of the regulatory sequences needed for geneexpression may vary between species or cell types, but shall in generalinclude, as necessary, 5′ non-transcribed and 5′ non-translatedsequences involved with the initiation of transcription and translationrespectively, such as a TATA box, capping sequence, CCAAT sequence, andthe like. Especially, such 5′ non-transcribed regulatory sequences willinclude a promoter region which includes a promoter sequence fortranscriptional control of the operably joined coding sequence.Regulatory sequences may also include enhancer sequences or upstreamactivator sequences as desired. The vectors of the invention mayoptionally include 5′ leader or signal sequences. The choice and designof an appropriate vector is within the ability and discretion of one ofordinary skill in the art.

Expression vectors containing all the necessary elements for expressionare commercially available and known to those skilled in the art. See,e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press, 1989. Cells aregenetically engineered by the introduction into the cells ofheterologous nucleic acid, usually DNA, molecules, encoding a biotinligase mutant. The heterologous nucleic acid molecules are placed underoperable control of transcriptional elements to permit the expression ofthe heterologous nucleic acid molecules in the host cell.

Preferred systems for mRNA expression in mammalian cells are those suchas pcDNA3.1 (available from Invitrogen, Carlsbad, Calif.) that contain aselectable marker such as a gene that confers G418 resistance (whichfacilitates the selection of stably transfected cell lines) and thehuman cytomegalovirus (CMV) enhancer-promoter sequences. Additionally,suitable for expression in primate or canine cell lines is the pCEP4vector (Invitrogen, Carlsbad, Calif.), which contains an Epstein Barrvirus (EBV) origin of replication, facilitating the maintenance ofplasmid as a multicopy extrachromosomal element. Another expressionvector is the pEF-BOS plasmid containing the promoter of polypeptideElongation Factor 1α, which stimulates efficiently transcription invitro. The plasmid is described by Mishizuma and Nagata (Nuc. Acids Res.18:5322, 1990), and its use in transfection experiments is disclosed by,for example, Demoulin (Mol. Cell. Biol. 16:4710–4716, 1996). Stillanother preferred expression vector is an adenovirus, described byStratford-Perricaudet, which is defective for E1 and E3 proteins (J.Clin. Invest. 90:626–630, 1992). The use of the adenovirus as anAdeno.P1A recombinant is disclosed by Warnier et al., in intradermalinjection in mice for immunization against P1A (Int. J. Cancer,67:303–310, 1996).

The invention also embraces so-called expression kits, which allow theartisan to prepare a desired expression vector or vectors. Suchexpression kits include at least separate portions of each of thepreviously discussed coding sequences. Other components may be added, asdesired, as long as the previously mentioned sequences, which arerequired, are included.

It will also be recognized that the invention embraces the use of theabove described, biotin ligase mutant encoding nucleic acid containingexpression vectors, to transfect host cells and cell lines, be theseprokaryotic (e.g., E. coli), or eukaryotic (e.g., rodent cells such asCHO cells, primate cells such as COS cells, Drosophila cells, Zebrafishcells, Xenopus cells, C. elegans cells, yeast expression systems andrecombinant baculovirus expression in insect cells). Especially usefulare mammalian cells such as human, mouse, hamster, pig, goat, primate,etc., from a wide variety of tissue types including primary cells andestablished cell lines.

Various methods of the invention also require expression of fusionproteins in vivo. The fusion proteins are generally recombinantlyproduced proteins that comprise the biotin ligase acceptor peptides.Such fusions can be made from virtually any protein and those ofordinary skill in the art will be familiar with such methods. Furtherconjugation methodology is also provided in U.S. Pat. Nos. 5,932,433;5,874,239 and 5,723,584.

In some instances, it may be desirable to place the biotin ligase mutantand possibly the fusion protein under the control of an induciblepromoter. An inducible promoter is one that is active in the presence(or absence) of a particular moiety. Accordingly, it is notconstitutively active. Examples of inducible promoters are known in theart and include the tetracycline responsive promoters and regulatorysequences such as tetracycline-inducible T7 promoter system, and hypoxiainducible systems (Hu et al. Mol Cell Biol. 2003December;23(24):9361–74). Other mechanisms for controlling expressionfrom a particular locus include the use of synthetic short interferingRNAs (siRNAs).

As used herein with respect to nucleic acids, the term “isolated” means:(i) amplified in vitro by, for example, polymerase chain reaction (PCR);(ii) recombinantly produced by cloning; (iii) purified, as by cleavageand gel separation; or (iv) synthesized by, for example, chemicalsynthesis. An isolated nucleic acid is one which is readily manipulableby recombinant DNA techniques well known in the art. Thus, a nucleotidesequence contained in a vector in which 5′ and 3′ restriction sites areknown or for which polymerase chain reaction (PCR) primer sequences havebeen disclosed is considered isolated but a nucleic acid sequenceexisting in its native state in its natural host is not. An isolatednucleic acid may be substantially purified, but need not be. Forexample, a nucleic acid that is isolated within a cloning or expressionvector is not pure in that it may comprise only a tiny percentage of thematerial in the cell in which it resides. Such a nucleic acid isisolated, however, as the term is used herein because it is readilymanipulable by standard techniques known to those of ordinary skill inthe art.

As used herein, a subject shall mean an organism such as an insect, ayeast cell, a worm, a fish, or a human or animal including but notlimited to a dog, cat, horse, cow, pig, sheep, goat, chicken, rodente.g., rats and mice, primate, e.g., monkey. Subjects include vertebrateand invertebrate species. Subjects can be house pets (e.g., dogs, cats,fish, etc.), agricultural stock animals (e.g., cows, horses, pigs,chickens, etc.), laboratory animals (e.g., mice, rats, rabbits, etc.),zoo animals (e.g., lions, giraffes, etc.), but are not so limited.

The compositions, as described above, are administered in effectiveamounts for labeling of the target proteins. The effective amount willdepend upon the mode of administration, the location of the cells beingtargeted, the amount of target protein present and the level of labelingdesired.

The methods of the invention, generally speaking, may be practiced usingany mode of administration that is medically acceptable, meaning anymode that produces effective levels of the active compounds withoutcausing clinically unacceptable adverse effects. A variety ofadministration routes are available including but not limited to oral,rectal, topical, nasal, intradermal, or parenteral routes. The term“parenteral” includes subcutaneous, intravenous, intramuscular, orinfusion.

When peptides are used, in certain embodiments one desirable route ofadministration is by pulmonary aerosol. Techniques for preparing aerosoldelivery systems containing peptides are well known to those of skill inthe art. Generally, such systems should utilize components which willnot significantly impair the biological properties of the peptides orproteins (see, for example, Sciarra and Cutie, “Aerosols,” inRemington's Pharmaceutical Sciences, 18th edition, 1990, pp 1694–1712;incorporated by reference). Those of skill in the art can readilydetermine the various parameters and conditions for producing protein orpeptide aerosols without resort to undue experimentation.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like. Lower doses will result from other forms ofadministration, such as intravenous administration. In the event that aresponse in a subject is insufficient at the initial doses applied,higher doses (or effectively higher doses by a different, more localizeddelivery route) may be employed to the extent that subject tolerancepermits. Multiple doses per day are contemplated to achieve appropriatesystemic levels of compounds.

The agents may be combined, optionally, with apharmaceutically-acceptable carrier. The term“pharmaceutically-acceptable carrier” as used herein means one or morecompatible solid or liquid filler, diluents or encapsulating substanceswhich are suitable for administration into a subject. The term “carrier”denotes an organic or inorganic ingredient, natural or synthetic, withwhich the active ingredient is combined to facilitate the application.The components of the pharmaceutical compositions also are capable ofbeing commingled with the molecules of the present invention, and witheach other, in a manner such that there is no interaction which wouldsubstantially impair the desired pharmaceutical efficacy.

The invention in other aspects includes pharmaceutical compositions.When administered, the pharmaceutical preparations of the invention areapplied in pharmaceutically-acceptable amounts and inpharmaceutically-acceptably compositions. Such preparations mayroutinely contain salt, buffering agents, preservatives, compatiblecarriers, and the like. When used in medicine, the salts should bepharmaceutically acceptable, but non-pharmaceutically acceptable saltsmay conveniently be used to prepare pharmaceutically-acceptable saltsthereof and are not excluded from the scope of the invention. Suchpharmacologically and pharmaceutically-acceptable salts include, but arenot limited to, those prepared from the following acids: hydrochloric,hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic,citric, formic, malonic, succinic, and the like. Also,pharmaceutically-acceptable salts can be prepared as alkaline metal oralkaline earth salts, such as sodium, potassium or calcium salts.

Various techniques may be employed for introducing nucleic acids of theinvention into cells, depending on whether the nucleic acids areintroduced in vitro or in vivo in a host. Such techniques includetransfection of nucleic acid-CaPO₄ precipitates, transfection of nucleicacids associated with DEAE, transfection with a retrovirus including thenucleic acid of interest, liposome mediated transfection, and the like.For certain uses, it is preferred to target the nucleic acid toparticular cells. In such instances, a vehicle used for delivering anucleic acid of the invention into a cell (e.g., a retrovirus, or othervirus; a liposome) can have a targeting molecule attached thereto. Forexample, a molecule such as an antibody specific for a surface membraneprotein on the target cell or a ligand for a receptor on the target cellcan be bound to or incorporated within the nucleic acid deliveryvehicle. For example, where liposomes are employed to deliver thenucleic acids of the invention, proteins which bind to a surfacemembrane protein associated with endocytosis may be incorporated intothe liposome formulation for targeting and/or to facilitate uptake. Suchproteins include capsid proteins or fragments thereof tropic for aparticular cell type, antibodies for proteins which undergointernalization in cycling, proteins that target intracellularlocalization and enhance intracellular half life, and the like.Polymeric delivery systems also have been used successfully to delivernucleic acids into cells, as is known by those skilled in the art. Suchsystems even permit oral delivery of nucleic acids.

Other delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of the labeling reagents. Many types of release deliverysystems are available and known to those of ordinary skill in the art.They include polymer base systems such as poly(lactide-glycolide),copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters,polyhydroxybutyric acid, and polyanhydrides. Microcapsules of theforegoing polymers containing drugs are described in, for example, U.S.Pat. No. 5,075,109. Delivery systems also include non-polymer systemsthat are: lipids including sterols such as cholesterol, cholesterolesters and fatty acids or neutral fats such as mono- di- andtri-glycerides; hydrogel release systems; sylastic systems; peptidebased systems; wax coatings; compressed tablets using conventionalbinders and excipients; partially fused implants; and the like. Specificexamples include, but are not limited to: (a) erosional systems in whichthe anti-inflammatory agent is contained in a form within a matrix suchas those described in U.S. Pat. Nos. 4,452,775, 4,667,014, 4,748,034 and5,239,660 and (b) diffusional systems in which an active componentpermeates at a controlled rate from a polymer such as described in U.S.Pat. Nos. 3,832,253, and 3,854,480.

A preferred delivery system of the invention is a colloidal dispersionsystem. Colloidal dispersion systems include lipid-based systemsincluding oil-in-water emulsions, micelles, mixed micelles, andliposomes. A preferred colloidal system of the invention is a liposome.Liposomes are artificial membrane vessels which are useful as a deliveryvector in vivo or in vitro. It has been shown that large unilamellarvessels (LUV), which range in size from 0.2–4.0 μm can encapsulate largemacromolecules. RNA, DNA, and intact virions can be encapsulated withinthe aqueous interior and be delivered to cells in a biologically activeform (Fraley, et al., Trends Biochem. Sci., (1981) 6:77). In order for aliposome to be an efficient gene transfer vector, one or more of thefollowing characteristics should be present: (1) encapsulation of thegene of interest at high efficiency with retention of biologicalactivity; (2) preferential and substantial binding to a target cell incomparison to non-target cells; (3) delivery of the aqueous contents ofthe vesicle to the target cell cytoplasm at high efficiency; and (4)accurate and effective expression of genetic information.

Liposomes may be targeted to a particular tissue by coupling theliposome to a specific ligand such as a monoclonal antibody, sugar,glycolipid, or protein. Liposomes are commercially available from GibcoBRL, for example, as LIPOFECTIN™ and LIPOFECTACE™, which are formed ofcationic lipids such as N-[1-(2, 3 dioleyloxy)-propyl]-N, N,N-trimethylammonium chloride (DOTMA) and dimethyl dioctadecylammoniumbromide (DDAB). Methods for making liposomes are well known in the artand have been described in many publications. Liposomes also have beenreviewed by Gregoriadis, G. in Trends in Biotechnology, (1985)3:235–241.

In one important embodiment, the preferred vehicle is a biocompatiblemicroparticle or implant that is suitable for implantation into themammalian recipient. Exemplary bioerodible implants that are useful inaccordance with this method are described in PCT Internationalapplication no. PCT/US/03307 (Publication No. WO 95/24929, entitled“Polymeric Gene Delivery System”). PCT/US/03307 describes abiocompatible, preferably biodegradable polymeric matrix for containingan exogenous gene under the control of an appropriate promoter. Thepolymeric matrix is used to achieve sustained release of the exogenousgene in the patient. In accordance with the instant invention, thefugetactic agents described herein are encapsulated or dispersed withinthe biocompatible, preferably biodegradable polymeric matrix disclosedin PCT/US/03307.

The polymeric matrix preferably is in the form of a microparticle suchas a microsphere (wherein an agent is dispersed throughout a solidpolymeric matrix) or a microcapsule (wherein an agent is stored in thecore of a polymeric shell). Other forms of the polymeric matrix forcontaining an agent include films, coatings, gels, implants, and stents.The size and composition of the polymeric matrix device is selected toresult in favorable release kinetics in the tissue into which the matrixis introduced. The size of the polymeric matrix further is selectedaccording to the method of delivery which is to be used. Preferably whenan aerosol route is used the polymeric matrix and agent are encompassedin a surfactant vehicle. The polymeric matrix composition can beselected.to have both favorable degradation rates and also to be formedof a material which is bioadhesive, to further increase theeffectiveness of transfer. The matrix composition also can be selectednot to degrade, but rather, to release by diffusion over an extendedperiod of time.

In another important embodiment the delivery system is a biocompatiblemicrosphere that is suitable for local, site-specific delivery. Suchmicrospheres are disclosed in Chickering et al., Biotech. And Bioeng.,(1996) 52:96–101 and Mathiowitz et al., Nature, (1997) 386:.410–414.

Both non-biodegradable and biodegradable polymeric matrices can be usedto deliver the agents of the invention to the subject. Biodegradablematrices are preferred. Such polymers may be natural or syntheticpolymers. Synthetic polymers are preferred. The polymer is selectedbased on the period of time over which release is desired, generally inthe order of a few hours to a year or longer. Typically, release over aperiod ranging from between a few hours and three to twelve months ismost desirable. The polymer optionally is in the form of a hydrogel thatcan absorb up to about 90% of its weight in water and further,optionally is cross-linked with multivalent ions or other polymers.

In general, agents are delivered using a bioerodible implant by way ofdiffusion, or more preferably, by degradation of the polymeric matrix.Exemplary synthetic polymers which can be used to form the biodegradabledelivery system include: polyamides, polycarbonates, polyalkylenes,polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates,polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, poly-vinylhalides, polyvinylpyrrolidone, polyglycolides, polysiloxanes,polyurethanes and co-polymers thereof, alkyl cellulose, hydroxyalkylcelluloses, cellulose ethers, cellulose esters, nitro celluloses,polymers of acrylic and methacrylic esters, methyl cellulose, ethylcellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose,hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate,cellulose acetate butyrate, cellulose acetate phthalate, carboxylethylcellulose, cellulose triacetate, cellulose sulphate sodium salt,poly(methyl methacrylate), poly(ethyl methacrylate),poly(butylmethacrylate), poly(isobutyl methacrylate),poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(laurylmethacrylate), poly(phenyl methacrylate), poly(methyl acrylate),poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecylacrylate), polyethylene, polypropylene, poly(ethylene glycol),poly(ethylene oxide), poly(ethylene terephthalate), poly(vinylalcohols), polyvinyl acetate, poly vinyl chloride, polystyrene,polyvinylpyrrolidone, and polymers of lactic acid and glycolic acid,polyanhydrides, poly(ortho)esters, poly(butiric acid), poly(valericacid), and poly(lactide-cocaprolactone), and natural polymers such asalginate and other polysaccharides including dextran and cellulose,collagen, chemical derivatives thereof (substitutions, additions ofchemical groups, for example, alkyl, alkylene, hydroxylations,oxidations, and other modifications routinely made by those skilled inthe art), albumin and other hydrophilic proteins, zein and otherprolamines and hydrophobic proteins, copolyrners and mixtures thereof.In general, these materials degrade either by enzymatic hydrolysis orexposure to water in vivo, by surface or bulk erosion.

Examples of non-biodegradable polymers include ethylene vinyl acetate,poly(meth)acrylic acid, polyamides, copolymers and mixtures thereof.

Bioadhesive polymers of particular interest include bioerodiblehydrogels described by H. S. Sawhney, C. P. Pathak and J. A. Hubell inMacromolecules, (1993) 26:581–587, the teachings of which areincorporated herein, polyhyaluronic acids, casein, gelatin, glutin,polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methylmethacrylates), poly(ethyl methacrylates), poly(butylmethacrylate),poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate),poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutylacrylate), and poly(octadecyl acrylate).

In addition, important embodiments of the invention include pump-basedhardware delivery systems, some of which are adapted for implantation.Such implantable pumps include controlled-release microchips. Apreferred controlled-release microchip is described in Santini, J T Jr.,et al., Nature, 1999, 397:335–338, the contents of which are expresslyincorporated herein by reference.

Use of a long-term sustained release implant may be particularlysuitable for treatment of chronic conditions. Long-term release, as usedherein, means that the implant is constructed and arranged to deliverytherapeutic levels of the active ingredient for at least 30 days, andpreferably 60 days. Long-term sustained release implants are well-knownto those of ordinary skill in the art and include some of the releasesystems described above.

The invention will be more fully understood by reference to thefollowing examples. These examples, however, are merely intended toillustrate the embodiments of the invention and are not to be construedto limit the scope of the invention.

EXAMPLES

Introduction

Many natural enzymes have evolved marked substrate specificity tofulfill their biological functions. One examples is E. coli enzymebiotin ligase (i.e., BirA) which participates in the transfer of CO₂from bicarbonate to organic acids to form various cellular metabolite.(Chapman-Smith et al. J. Nutr. 129:477S–484S, 1999.) It has only onenatural substrate in bacteria: the biotin carboxyl carrier protein(BCCP), which it biotinylates at lysine 122 to prepare it forcarboxylation by bicarbonate. Schatz et al. used peptide panning toidentify a minimal, 13-amino acid peptide sequence that could berecognized and enzymatically biotinylated by BirA: LNDIFEAQKIEWH (SEQ IDNO:4), where the biotinylated lysine is underlined. (Schatz et al.Biotechnology 11:1138–1143, 1993; Beckett et al. Protein Sci. 8:921–929,1999.) Purified BirA and cloning vectors for introducing thismodification sequence, called “Avi-Tag™,” onto proteins of interest forsite-specific biotinylation in vitro or in living bacteria arecommercially available. (Avidity, Boulder, Colo. ) Recently, Stroubouliset al. reported that BirA could also be used to efficiently andspecifically biotinylate Avi-tagged proteins in mammalian cells. (deBoer et al. PNAS 100:7480–7485, 2003.) The E. coli BirA does notbiotinylate any endogenous mammalian proteins, and the mammaliancounterpart of BirA does not biotinylate the Avi-Tag.

According to the invention, the biotin binding pocket of BirA wasre-engineered to accommodate a range of small-molecule probes other thanbiotin. Mutants of BirA that can efficiently catalyze the attachment ofvarious small molecule probes (i.e., biotin analogs) to Avi-taggedprotein substrates in vitro and in mammalian cells have been developed.The remaining domains of the protein were left intact, including theresidues important for ATP binding, peptide substrate binding, andcatalysis. The re-engineered BirA is useful for targeting small moleculedetectable (e.g., fluorescent) probes to specific proteins in livecells.

i. Rational Mutation of Biotin Ligase (BirA) Active Site to Relax itsSpecificity for Biotin.

The published crystallographic and biochemical data were used to designa panel of biotin ligase mutants with altered biotin binding sites. Thetwo co-crystal structures of 33.5 kD BirA complexed to biotin andbiotinylated lysine show a binding pocket composed of both hydrophobicresidues (186, 204, 206) which contact the thiophene ring of biotin, andhydrophilic residues (89, 90, 112, 115, 116, 118, 123) which formhydrogen bonds to the carbonyl and ureido nitrogen groups. (Wilson etal. PNAS 89:9257–9261, 1992 and Weaver et al. PNAS 98:6045–6050, 2001.)Mutagenesis studies have also identified several “second-shell” aminoacids (83, 107, 142, 189, 207) important for biotin affinity.

By inspecting the 2.4 Å BirA-biotin co-crystal structure, several keyresidues were identified that are directly in contact with the bicycliccore of biotin. These residues were changed individually by mutagenesisto enlarge the biotin binding site. Two different probes, an N-ketonebiotin analog and an N-alkyne biotin analog (FIG. 1B), were found toeffectively compete against biotin for binding to two BirA mutants—T90Gand T90G/N91S, respectively, as shown in a competitive inhibition assayusing ³H-labeled biotin (Table 1). The N-ketone and N-alkyne probes bothbear substitutions on the trans ureido nitrogen of biotin, whichdirectly interferes with the T90 residue. Reduction of the T90 sidechain to a proton (e.g., glycine) makes room for these ketone and alkynemoieties, allowing them to fit into the biotin binding pocket. In thecase of the alkyne probe, which has a slightly different geometry thanthe ketone, additional space generated by changing N91 to serine isrequired. These results show that the BirA structure is amenable toreengineering and that certain non-naturally occurring biotin analogs(i.e., structurally biotin-like molecules) can be accommodated in thebiotin binding site after careful mutagenesis.

TABLE 1 Incorporation of N-ketone and N-alkylene biotin analogsx by theBirA mutants T90G and T90G/N91S, respectively, as measured in acompetitive inhibition assay with ³H-labeled biotin. % Inhibition of N-% Inhibition of Mutant N-Ketone ³H-biotin incorporation Mutant Alkylene³H-biotin incorporation WT 0 0% WT 0   0% WT 4 mM <50% WT 2 mM   5%G115A 4 mM <50% Y132A 2 mM   0% T90G/N91S 4 mM 80% G115A 2 mM   0% T90V4 mM <50% Q112M 2 mM 1.6% T90A 4 mM <50% T90A 2 mM   0% T90G 4 mM 100%T90A/N91A 2 mM   0% T90A/N91L 2 mMN   0% T90V 2 mM   0% T90V/N91L 2 mM1.6% T90G 2 mM  12% T90G/N91S 2 mM  77%

Ketones and alkynes are useful functional groups to incorporate intoproteins because they can be subsequently ligated in bio-orthogonalconjugation reactions to hydrazide or azide-derivatized fluorophores.For example, specific ketone-hydrazide ligation has been reported byBertozzi et al. on the surface of live mammalian cells and in cellextracts, and alkyne-azide ligation via a [3+2] cycloaddition reactionhas been reported on Cowpea mosaic virus coat proteins and on thesurface of bacteria. (Mahal et al. Science 276:1125–1128, 1997; Wang etal. J. Am. Chem. Soc. 125:3192–3193, 2003; Link et al. J. Am. Chem. Soc.125:11164–11165, 2003.)

T90G has therefore been identified according to the invention as animportant residue for accommodating N-substituted biotin analog typeprobes. Additional biotin analogs can be tested for incorporation usinga panel of seventeen rationally-designed BirA point mutants: T90G, T90V,T90A, T90G/N91S, T90G/N91G, T90A/N91A, T90A/N91L, T90V/N91L, C107G,Q112G, Q112M, G115A, Y132A, Y132G, V189G, S134G, and I207S. Many of thecontacts with biotin are via side chains rather than backbone elements,indicating an opportunity to carve out considerable space to accommodatenon-naturally occurring probes. Also, there is a large water-filledchannel above the ureido moiety of biotin that appears wide enough toaccommodate even larger structures (e.g., coumarin and fluorescein).

Mutant BirA can also be expressed, purified and tested in 96-wellplates. The western blot assays described herein for analyzing probeincorporation have already been adapted to a plate format for mediumthroughput.

In addition, amino acids in the biotin binding site are beingcomputationally randomized and subsequently analyzed using particularalgorithms to search for protein sequences that bind to various biotinanalogs with high affinity.

Biotin analog incorporation can be determined using a variety of assaysincluding but not limited to (1) inhibition of ³H-biotin incorporation,(2) western blot detection of unnatural probe conjugation to cyanfluorescent protein (CFP) bearing a C-terminal Avi-Tag, (3) MALDImass-spectrometric detection of probe attachment to an Avi-Tag peptidesubstrate, and (4) HPLC. In the first of these assays, biotin analogcandidates and biotin are incubated together with the biotin ligasemutant and the acceptor peptide. Decreases in incorporation ofradioactivity are indicative of a biotin analog that competeseffectively with biotin for the biotin ligase mutant activity. In thesecond of these assays, biotin analog conjugation to an acceptor peptideis indicated by the use of antibodies specific for the biotin analog ora label conjugated thereto (e.g., an anti-FLAG antibody or ananti-fluorophore antibody). In the third assay, differences in themolecular weight of the acceptor peptide are indicative of incorporationof the biotin analog. In the last of these assay, acceptor peptides withlonger retention times are indicative of biotin analog incorporation.

ii. Synthesis of Biotin Analogs with Unique Biophysical or ChemicalProperties Such as Fluorescence.

A range of probes for both in vitro and cellular applications wassynthesized and tested against the panel of BirA mutants. Synthesispathways are illustrated in FIGS. 4 and 5. A fluorophore similar inshape and size to the biotin ring system, 7-nitrobenz-2-oxa-1,3-diazole(NBD), has been conjugated to γ-aminobutyric acid (GABA) to yieldNBD-GABA biotin analog (FIG. 1B). Initial analysis of NBD-GABA indicatesthat it has a low fluorescence quantum yield in water and shortexcitation wavelength (˜340 nm), making it suboptimal for live cellimaging. However, its high sensitivity to variations in localenvironment make it highly useful as an in vitro biophysical probe.

Ketone biotin analog (FIG. 1B) is not by itself a biophysical probe, butonce conjugated to a protein of interest, can serve as a chemical handlefor selective derivatization with hydrazine or alkoxyamine-bearingprobes (FIG. 2). (Cornish et al. J. Am. Chem. Soc. 118:8150–8151, 1996;and Mahal et al. Science 276:1125–1128, 1997.) This chemistry isspecific for the introduced ketone over other functionalities present onmammalian cell surfaces. (Mahal et al. Science 276:1125–1128, 1997.)Inside a cell, however, hydrazides must be prevented from coupling toketone and aldehyde carbonyls of carbohydrates and natural cofactors.This selectivity may be achieved through multivalency (e.g., twomodification sequences may be linked in tandem to a protein of interest,and a bis-functionalized fluorophore with two appropriately-spacedhydrazide groups would have a thermodynamic preference for the targetprotein over endogenous carbonyl compounds). A heterodivalentinteraction may also be achieved by introducing a cysteine residue nearthe lysine modification site in the BirA target sequence and a probebearing both a hydrazine moiety and a thiol group would be able to forma hydrazone-disulfide macrocyclic adduct.

Two other biotin derivatives that would similarly introduce chemicallyunique handles for subsequent modification by probes are shown in FIG.1B. The Staudinger reaction between an azide and a phosphine has beenreported in live cells, as has complexation between fluorescein-arsenicand a tetrathiol moiety. (Saxon et al. Science 287:2007–2010, 2000 andGriffin et al. Science 281:269–272, 1998.)

Lastly, probes that provide readouts other than fluorescence, or alterprotein function, can also be used with the panel of BirA mutants. Suchprobes may include MRI contrast reagents, PET labels, phosphorescent orluminescent tags, singlet-oxygen generators for electron microscopystaining, heavy atoms, photoactivatable crosslinkers (e.g.,benzophenones), photoswitches (e.g., azobenzenes), and photocagedlabels.

iii. Screening of BirA Mutants for Ability to Conjugate Biotin Analogsto a Lysine Side Chain within a 13-Amino Acid Consensus Sequence.

Wild-type BirA and several of the mutants listed herein have beenexpressed and purified. Screening of these enzymes for ability toconjugate NBD-GABA biotin analog to a cyan fluorescent protein (CFP)substrate with a C-terminal 13-amino acid modification sequence (“CFP-AviTag™”) is detected using anti-DNP (dinitrophenyl) antibody (MolecularProbes) in a Western blot format. To detect ketone conjugation,enzymatic reaction mixtures are treated with fluorescein hydrazide,subjected to gel filtration or Ni-NTA purification to separate CFP-AviTag™ (which bears an N-terminal His₆ tag) from unreacted dye, andassayed by fluorimetry. Other biotin analogs are screened in a similarmanner.

iv. Generation of Further BirA Mutants Using a Phase Library Approach.

Further BirA mutants can be generated using phage display and mammaliancell FACS (fluorescence activated cell sorting). Some of the biotinanalogs described herein are sufficiently structurally similar to biotinthat they are likely to be accepted by both wild-type BirA or one of thesingle-point mutants. In some embodiments, wild type BirA may havereduced affinity for the biotin analog however.

For other analogs, more extensive active-site reengineering is required.Instead of screening mutants one-by-one, a more efficient approach usesdirected evolution techniques to select suitable BirA mutants from largelibraries. Neri et al. have reported the successful display of activewild type BirA on the surface of bacteriophage and developed an in vitroselection scheme for separating active enzymes from inactive ones.(Heinis et al. Protein Engineering, 14:1043–1052, 2001. ) A library ofBirA mutants was designed, using the crystal structures and biochemicalreports as guides, to be displayed on the surface of bacteriophage. Toenrich for suitable BirA mutants, anti-fluorophore antibodies such asanti-DNP or anti-fluorescein as shown in FIG. 3A are used. The BirAlibrary can be DNA-shuffled between selection rounds to increasediversity and hasten consensus towards active BirA mutants. Negativeselections against mutants still capable of transferring biotin can alsobe implemented using streptavidin beads.

A phage display-based selection system for identification of BirAmutants capable of catalyzing biotin analog conjugation to an Avi-Tagpeptide has been developed. The selection uses a calmodulin-M13 strategy(Heinis et al. Protein Engineering, 14:1043–1052, 2001) to anchor theAvi-Tag peptide substrate to the protein coat of each phage molecule.The BirA library is joined to calmodulin and this fusion protein isdisplayed on the phage coat protein pIII. Model selections havedemonstrated that phage displaying wild-type BirA can be enriched overphage displaying a dead mutant (G115S) by 42-fold in one round ofselection. It has also been shown that phage molecules chemicallylabeled with the ketone probe or with the NDB probe shown above can beenriched over mock-labeled phage by 14-fold (using antibodies againstNBD or the hydrazide-containing epitope ligated to the ketone).

Libraries that are biased for particular mutations are alsocontemplated. For example, libraries that are based on a T90G amino acidsubstitution are a starting template for N-substituted biotin analogs.In other instances, the library can be randomized at seven positionsnear biotin (i.e., 90, 91, 112, 115, 116, 132 and 188). This library hasa size of 1.3×10⁹.

Selection in cells is accomplished by co-transfection with a BirAconsensus substrate sequence (i.e., the acceptor peptide) fused to cyanfluorescent protein (CFP), which displays fluorescence resonance energytransfer (FRET) to any successfully incorporated probe, allowing FACSselection. The advantage of labeling an already-fluorescent protein isthat non-specific labeling of endogenous proteins will not result in aFRET signal. Labeling specificity can be measured using the ratio ofFRET to total fluorescence.

v. In Vivo Site-specific Labeling Methodology.

BirA mutants that perform well in vitro are subsequently screened foractivity in mammalian cells. First, BirA mutants that specifically labelat the target sequence, thereby discriminating against all endogenousmammalian proteins, are selected. E. coli BirA has naturally evolved asignificant degree of peptide specificity in its bacterial context.Peptide panning reportedly has shown that the substrate specificities ofE. coli BirA and yeast biotin ligase are non-overlapping. (Kiick et al.PNAS 99:19–24, 2000.) To test whether this orthogonality is also foundin the desired mammalian intracellular milieu, mammalian cells aretransfected with the BirA mutant nucleic acid sequence as describedherein and any undesired modification of endogenous mammalian proteinsis detected by Western blot. If background labeling is observed, thenthe peptide substrate specificity of the enzyme will be targeted forre-engineering using the FRET/total fluorescence ratio readout outlinedherein.

Second, biotin analogs must permeate cells and tissues readily. Biotinis too polar to cross the plasma membrane and requires a transporterprotein. The methyl ester of biotin, however, crosses membranes readilyand is hydrolyzed to biotin intracellularly by endogenous esterases. Themembrane permeance of biotin analogs can be tested, using fluorescenceas the readout. Probes that are too polar to cross the membrane will bederivatized to their ester form.

Third, mutant BirA expression level must be high enough that targetproteins will be labeled efficiently. However, overexpression can leadto toxicity. The selection strategy in some instances would favor astable cell line that expresses the mutant BirA consistently and atmoderate levels. Alternatively, the gene encoding mutant BirA is placedunder control of an inducible promoter and enzyme expression is turnedon only when needed.

Finally, the unconjugated probe must be washed out in order to minimizebackground staining (except for fluorogenic compounds such as FlAsH).Repeated washing with fresh growth media may be sufficient in manycases. In others, addition of probe-specific quenching reagents may behelpful for “stickier” small molecules. Examples of probe-specificquenching reagents include ethandithiol (used for example to removeunbound labels in fluorescein arsenic labeling).

vi. Application to the Study of PI3-Kinase Activation Patterns in 3T3-L1Adipocytes, or Fat Storage Cells, in Response to PDGF (Platelet-derivedGrowth Factor) and Insulin Stimulation.

As an example, mutant BirA can be applied to the study of PI3-kinaseactivation in 3T3-L1 adipocytes. These adipocytes display a membraneruffling response to PDGF and a glucose transport response to insulin,both mediated by PI3-kinase stimulation. These differing downstreameffects may result, according to one hypothesis, from activation ofspatially and/or temporally separate pools of PI3-kinase. To test this,a two-tag FRET system is constructed by enzymatically labeling thecatalytic and regulatory subunits of PI3-kinase inside cells. Smallfluorophores should perturb the system far less than fluorescentproteins such as GFP. This system allows measurement of PI3-kinaseactivation in real time and at subcellular resolution after insulin orPDGF stimulation.

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EQUIVALENTS

It should be understood that the preceding is merely a detaileddescription of certain embodiments. It therefore should be apparent tothose of ordinary skill in the art that various modifications andequivalents can be made without departing from the spirit and scope ofthe invention, and with no more than routine experimentation. It isintended to encompass all such modifications and equivalents within thescope of the appended claims.

All references, patents and patent applications that are recited in thisapplication are incorporated by reference herein in their entirety.

1. A method for labeling a target protein comprising contacting a fusionprotein with a biotin analog, and allowing sufficient time for thebiotin analog to be conjugated to the fusion protein via an acceptorpeptide, in the presence of a biotin ligase mutant, wherein the fusionprotein is a fusion of the target protein and the acceptor peptide, andwherein the biotin ligase mutant is a mutant of SEQ ID NO: 1 andcomprises one or more amino acid substitutions selected from the groupconsisting of T90G, T90V, T90A, N91S, C107G, Q112M, G115A, Y132G, Y132A,S134G, V189G and I207S.
 2. The method of claim 1, wherein the biotinanalog comprises an aliphatic carboxylic acid tail.
 3. The method ofclaim 1, wherein the biotin analog comprises a substitution at atrans-ureido nitrogen (N) of biotin.
 4. The method of claim 1, whereinthe biotin analog is selected from the group consisting of an N-ketonebiotin analog, a ketone biotin analog, an N-azide biotin analog, anazide biotin analog, an N-acyl azide biotin analog, an NBD-GABA biotinanalog, a 1,2-diamine biotin analog, an N-alkyne biotin analog and atetrathiol biotin analog.
 5. The method of claim 1, wherein the targetprotein is a cell surface protein.
 6. The method of claim 1, wherein thefusion protein is in a cell.
 7. The method of claim 6, wherein the cellexpresses the biotin ligase mutant.
 8. The method of claim 6, whereinthe cell is a eukaryotic cell.
 9. The method of claim 6, wherein thecell is a bacterial cell.
 10. The method of claim 8, wherein theeukaryotic cell is a mammalian cell, a Drosophila cell, a Zebrafishcell, a Xenopus cell, a yeast cell or a C. elegans cell.
 11. The methodof claim 1, wherein the acceptor peptide comprises an amino acidsequence of SEQ ID NO:
 4. 12. The method of claim 1, wherein theacceptor peptide comprises an amino acid sequence of SEQ ID NO:
 5. 13.The method of claim 1, wherein the acceptor peptide is N- or C-terminally fused to the target protein.
 14. The method of claim 1,wherein the biotin analog is N-ketone biotin analog.
 15. The method ofclaim 1, wherein the biotin ligase mutant has an amino acid of SEQ IDNO:
 6. 16. The method of claim 1, wherein the biotin ligase mutantcomprises amino acid substitutions of T90G and N91S.
 17. The method ofclaim 16, wherein the biotin analog is N-alkyne biotin analog.
 18. Themethod of claim 16, wherein the biotin ligase mutant has an amino acidsequence of SEQ ID NO:
 7. 19. The method of claim 1, wherein the methodis performed in a cell free environment.
 20. The method of claim 1,wherein the method is performed in a cell.
 21. The method of claim 1,wherein the method is performed in a subject.
 22. The method of claim 1,wherein the acceptor peptide is fused to the target protein via acleavable bond or linker.